PROJECT SUMMARY Type 1 diabetes (T1D) is a complex disease mediated by T cell entry into pancreatic islets and the subsequent destruction of insulin-producing ? cells. Regulatory T cells (Tregs) are a critical inhibitory population that prevents autoimmunity in most individuals, but ultimately fails to prevent disease onset in patients with T1D. However, the mechanistic basis for this ?Treg insufficiency? remains poorly understood. We have recently identified a neuropilin-1:semaphorin-4a (Nrp1:Sema4a) axis that is required to maintain Treg stability, survival and function in mouse models of human cancer and inflammatory bowel disease. Treg-restricted loss of this pathway in mice leads to profound resistance to tumor growth, due to an inability to restrain anti-tumor immunity, but did not lead to loss of homeostatic Treg function that might have led to systemic inflammation and autoimmunity. This suggested that Tregs only require the Nrp1 pathway in inflammatory lesions, although it is not known if this pathway is used by Tregs in autoimmune diabetes. Surprisingly, while Treg-restricted loss of Nrp1 did not impact autoimmune diabetes, but enhanced Nrp1 signaling induced by ectopic ligand expression on islet ? cells reduced diabetes by ~50%. Further preliminary analysis of intra-islet Tregs revealed a progressive and selective loss of Nrp1, which limited the capacity of Tregs to use this pathway. In this competitive renewal application, we will determine if the Nrp1 pathway impacts autoimmune diabetes, and if enforced utilization limits Treg insufficiency and autoimmune diabetes. AIM 1: On the molecular basis of Treg insufficiency in autoimmune diabetes. In this Aim, we will ask: (A) Does enhanced intra-islet Nrp1-Treg ligation mediate protection from autoimmune diabetes? (B) What is the functional impact of Nrp1 loss on intra-islet Treg function? (C) What is the mechanism of Nrp1 shedding? (D) What drives Nrp1 shedding? AIM 2: Approaches to negate Treg insufficiency in autoimmune diabetes. In this Aim, we will ask: (A) Can Treg function be enhanced by nanoparticle-mediated delivery in vivo? (B) Can Treg function be enhanced in vivo using HSV-mediated gene delivery? This project will have a significant impact on our understanding of the mechanisms that underlie Treg insufficiency in autoimmune diabetes, and determine if two translational proof-of-concept approaches can boost Treg function/survival and limit autoimmune diabetes. Given that we discovered and elucidated the Nrp1 pathway and its physiological impact on Treg function/survival, and have many unique tools to probe its function, we are in the best position to conduct this research.